Reverse transcription is the process by which a retrovirus such as HIV-1 converts its single-stranded (ss) RNA genome into a double-stranded DNA copy that is integrated into host chromosomal DNA. This process is complex and is catalyzed by the virion-associated enzyme, reverse transcriptase (RT). However, another viral protein, the nucleocapsid protein (NC), is also required for efficient and specific viral DNA synthesis. (A) We study the mechanistic basis for NC activity. HIV-1 NC is a small (7 kDa), basic nucleic acid binding protein with two zinc fingers (ZFs), each containing the invariant CCHC zinc-coordinating motifs. It is also a nucleic acid chaperone: NC facilitates remodeling of nucleic acid structures so that the most thermodynamically stable conformations are formed. This property is critical for promoting initiation of reverse transcription by the host tRNALys3 primer and the two strand transfer events that are needed for synthesis of full-length minus- and plus-strand viral DNA. (i) Recent studies have focused on the nucleic acid chaperone activity of HIV-1 NC when it is embedded in Gag (the precursor to the viral structural proteins). Assays measuring initiation of minus-strand DNA synthesis, i.e., synthesis of (-) strong-stop DNA, demonstrate that Gag promotes tRNALys3 annealing to viral RNA and subsequent RT-catalyzed elongation of the annealed primer. However, Gag has 4-fold less activity than NC at optimum concentrations for each protein. At high concentrations of Gag, tRNA extension is reduced to background level, providing additional evidence for the ability of Gag to act as a roadblock to RT-catalyzed DNA elongation. This indicates that during virus infection, Gag must be cleaved by the viral protease to mature NC prior to the extension reaction. To determine whether the ZFs in the NC domain of Gag contribute to chaperone function, we used a genetic approach. Gag ZF deletion mutants have no activity in the initiation assay and His to Cys point mutants exhibit 2- to 3-fold lower activity than wild-type (WT) Gag. This shows that zinc coordination is required for Gag-facilitated initiation. In contrast, the minus-strand transfer activity of the Gag ZF mutants is very similar to that of WT. This observation raises the possibility that the positively charged residues in the matrix domain, which lies in close proximity to NC when Gag is in solution, may promote strand transfer via an aggregation-driven pathway. We are currently investigating the activity of NC's immediate precursor proteins, NC9 (NC plus SP1) and NC15 (NC9 plus p6) in minus-strand initiation and strand transfer assays. (ii) During synthesis of (-) strong-stop DNA, the RNase H activity of RT cleaves the viral RNA template, generating small 5-prime terminal RNA fragments that remain annealed to the DNA. Unless these fragments are removed, the minus-strand transfer reaction, required for (-) SSDNA elongation, cannot proceed. We hypothesized that fragment removal could be facilitated by NC destabilization of the short duplex and/or by RNase H cleavage. To test this hypothesis, we used an NC-dependent system that models minus-strand transfer. We showed that that the presence of short terminal fragments pre-annealed to (-) SSDNA has no impact on strand transfer, implying efficient fragment removal. Moreover, in reactions with an RNase H-minus RT mutant, NC alone is able to facilitate fragment removal, albeit less efficiently than in the presence of both RNase H and NC. Results obtained from novel gel-mobility shift and FRET assays, which each directly measure RNA fragment release from a duplex in the absence of DNA synthesis, demonstrated that the architectural integrity of NC's ZF domains is absolutely required for this reaction. These findings are in excellent agreement with our earlier studies of the tRNA removal step in plus-strand transfer (Wu et al. J.Virol. 73:4794-4805, 1999) and the ability of NC to block mispriming during initiation of plus-strand DNA synthesis (Post et al. NAR 37:1755-1766, 2009). Thus, HIV-1 uses a common mechanism for the three RNA removal reactions required for successful reverse transcription. (B) Our interest in host proteins that affect HIV-1 reverse transcription and replication has led us to investigate the activities of human APOBEC3 (A3) proteins, a family of ssDNA deoxcytidine deaminases that play an important role in the cellular immune response to viral pathogens. Currently, we are studying the human A3A protein, which degrades foreign DNA, blocks HIV-1 replication in myeloid cells, and inhibits retrotransposition of LINE-1 elements, a class of mobile genetic elements. These elements are potentially detrimental to the human genome, since they can insert into coding regions of functional genes. To test the role of A3A deaminase activity in the inhibition of LINE-1 retrotransposition, we performed cell-based assays with A3A WT and mutant constructs and correlated the levels of retrotransposition with catalytic activity. In general, mutants defective in deamination were also found to exhibit less anti-retrotransposition activity, suggesting a correlation between these two activities. To obtain further insights into the molecular properties of A3A, we are using highly purified recombinant protein to study its binding to nucleic acids as well as the kinetics and specificity of deamination. A3A is a slightly acidic protein and the apparent Kd values for binding are in the micromolar range. However, it is a potent deaminase and under our assay conditions, a reaction containing as little as 200 nM A3A has a half-life of 8.4 sec. Our collaborators, who are structural biologists, have recently determined the A3A solution structure by NMR. The overall fold is similar, though not identical to that of the catalytic C-terminal domain of A3G. The A3A structural work defines the interface that is crucial for interaction with the ssDNA substrate and complements our biochemical and biological studies. (C) Our laboratory has been investigating the role of the HIV-1 capsid protein (CA) in early postentry events, a stage in the infectious process that is still not completely understood. We have focused on the biological function of the flexible peptide, known as the interdomain linker, which connects the N- and C-terminal domains of CA. We made single Ala or Leu substitutions in each of the five linker residues (CA residues 146-150) and in the two flanking residues. Although all of the mutants produce virus particles, only two mutants (S146A and T148A) have infectivity in a single-cycle replication assay. The lack of infectivity of three other mutants (Y145A, I150A, and L151A) is correlated with defects in core morphology and stability, inability of purified CA proteins to assemble into a recognizable structure in vitro, as well as abrogation of viral DNA synthesis. Interestingly, two additional mutants (P147L and S149A), while poorly infectious, display an attenuated phenotype and surprisingly, their infectivity is rescued when env-minus virions are pseudotyped with the vesicular stomatitis envelope glycoprotein. Moreover, despite having unstable cores, these mutants (i) synthesize viral DNA in infected cells, although less efficiently than WT virus, (ii) assemble a significant number of viral cores with seemingly normal architecture, and (iii) assemble tubular structures in vitro that resemble WT assemblies. Taken together, our findings demonstrate that the HIV-1 interdomain linker region is a critical determinant of proper core assembly and stability.